Treatment method for cancer

ABSTRACT

The in vivo chemotherapeutic treatment of cancer cells in a living animal is improved by first administering to the animal, a compound which inhibits normal cell proliferation while promoting malignant cell proliferation, specifically a potent antagonist selective for intracellular histamine receptors, in an amount sufficient to inhibit the binding of intracellular histamine to the receptors in normal and malignant cells. An enhanced toxic effect on the cancer cells from the chemotherapeutic agent is obtained while any adverse effect of the chemotherapeutic agent on normal cells, particularly bone marrow and gastro-intestinal cells, is inhibited. In addition, long term continuous administration of the antagonist following administration of the chemotherapeutic agent results in at least amelioration of adverse side effects of chemotherapy on normal bone marrow and gastro-intestinal cells. The treatment of cancer cells using DPPE in combination with the chemotherapeutic agents specifically illustrates the invention.

REFERENCE TO RELATED APPLICATION

This application is a division of U.S. patent application Ser. No.08/082,785 filed Jun. 28, 1993, which itself is a continuation-in-partof my U.S. patent application Ser. No. 711,975 filed Jun. 7, 1991, nowabandoned, which itself is a continuation-in-part of U.S. patentapplication Ser. No. 627,863 filed Dec. 17, 1990 (now abandoned).

FIELD OF INVENTION

The present invention relates to the improved treatment of cancer inanimals, including humans, using chemotherapeutic agents.

BACKGROUND TO THE INVENTION

One of the major chemotherapeutic treatments is that of malignant growth(cancer) in humans. The objective of chemotherapy is the totalextermination of clonogenic tumor or malignant cells, with minimaldamage to the patient. However, one of the major limitations of thechemotherapeutic approach for managing human cancer is the generalinability of anticancer drugs to discriminate between normal andtumorous cells. Anti-neoplastic agents have the lowest therapeuticindicies of any class of drugs used in humans and hence producesignificant and potentially life-threatening toxicities. Certaincommonly-used anti-neoplastic agents have unique and acute toxicitiesfor specific tissues. For example, the vinca alkaloids possesssignificant toxicity for nervous tissues, while adriamycin has specifictoxicity for heart tissue and bleomycin has for lung tissue. In general,almost all members of the major categories of anti-neoplastic agentshave considerable toxicities for normal cells of gastrointestinal,epidermal and myelopoietic tissues.

Generally, the dose-limiting consideration for chemical management ofcancer in humans is the toxicity that anti-neoplastic agents have forthe pluripotent stem cells of myelopoietic tissue. This toxicity arisesfrom the fact that most anticancer drugs function preferentially againstproliferating cells but with no significant capacity to discriminatebetween cycling normal and cycling tumor tissues.

Attempts have been made to confer specificity upon presently-availablechemotherapeutic agents. In Anticancer Research 6:451 to 464 (1986),Robert C. Warrington describes certain in vitro and in vivo experimentsdemonstrating the achievement of improvements in both the specificityand efficacy of a number of commonly-used anticancer drugs by usingthese agents in combination with L-histidinol. L-histidinol is astructural analogue of the essential amino acid, histidine, in which theα-carboxyl group has been reduced to a primary alcohol, and is aprecursor of histamine. In the work presented by Warrington in thispaper, L-histidinol was found in mice to be effective at doses ofapproximately 1000 mg/kg (4000 mg/M²) of tissue administered five hoursor more prior to the chemotherapeutic agent.

SUMMARY OF INVENTION

It now surprisingly has been found that, if a potent antagonistselective for a recently-discovered intracellular histamine receptor,designated H_(IC), different from traditional histamine receptorsclassified as H₁, H₂ or H₃, is administered to a living animal havingcancer, then the specificity and efficacy of chemotherapeutic agents forcancer cells is improved. By employing a potent and selective antagonistto inhibit the binding of intracellular histamine, the improved effectis obtained at significantly lower dosage levels administered for asignificantly shorter period of time prior to administration of thechemotherapeutic agent than is shown in the Warrington work referred toabove.

The present invention is broadly applicable to the treatment ofmalignant cells in a living animal where the administration ofchemotherapeutic agents normally adversely affects the normal (i.e.non-malignant) cells in the animal. By first administering to the animala potent antagonist selective for intracellular histamine receptors inan amount sufficient to inhibit binding of intracellular histamine innormal cells at the intracellular histamine binding site, thespecificity and efficacy of subsequently administered therapeutic agentson malignant cell is improved.

Accordingly, in one aspect of the present invention, there is provided amethod for the treatment of cancer cells in a living animal, whichcomprises (a) administering to the animal a potent antagonist selectivefor intracellular histamine receptors in an amount sufficient to inhibitthe binding of intracellular histamine in normal and malignant cells,and (b) subsequently administering to the animal at least onechemotherapeutic agent in an amount toxic for the cancer cells, wherebyan enhanced toxic effect on the cancer cells from the at least onechemotherapeutic agent is obtained while, depending on the dose of theintracellular histamine antagonist, adverse effects of the at least onechemotherapeutic agent on normal cells, particularly bone-marrow orgastro-intestinal cells is significantly ameliorated.

It has further been found that, if a patient being treated with achemotherapeutic agent also is given an IV infusion of a low dose of apotent antagonist selective for intracellular histamine receptors over aperiod of from about 6 to about 72 hours after administration of thechemotherapeutic agent, then the gastro-intestinal side effectsgenerally associated with chemotherapy, namely nausea, vomiting,anorexia and stomatitis, are at least ameliorated and often prevented.

Accordingly, in another aspect of the present invention, there isprovided a method for the treatment of cancer cells, which comprises (a)administering to the animal at least one chemotherapeutic agent in anamount toxic for the cancer cells, and (b) administering to the animalfor a period of up to about seventy-two hours a potent antagonistselective for intracellular histamine receptors in an amount sufficientto inhibit the binding of intracellular histamine in normal cells,whereby the side effects of administration of the chemotherapeutic agentare at least ameliorated.

In the application of the present invention to human beings, thematerials generally are administered by intravenous infusion. In onepreferred procedure, a solution of the potent antagonist is administeredto the patient over the desired period of time prior to administrationof the chemotherapeutic agent, a solution of the chemotherapeutic agentin combination with the potent antagonist then is administered for theperiod of time of administration of the chemotherapeutic agent, and asolution of the potent antagonist thereafter is administered to thepatient for the desired period to ameliorate side effects from thechemotherapeutic agent administration.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1 to 14 are graphical representations of test data generated incertain experiments set forth in the Examples below.

GENERAL DESCRIPTION OF INVENTION

In the present invention, any compound which is a potent antagonist ofhistamine binding at the intracellular histamine receptor is useful andis administered in an amount sufficient to inhibit the binding ofintracellular histamine at the intracellular binding site (H_(IC)) innormal cells. Such compounds generally exhibit a pKi of at least about5, preferably at least about 5.5.

Specific potent compounds which are useful in the present invention arediphenyl compounds of the formula: ##STR1## wherein X and Y are eachfluorine, chlorine or bromine, Z is an alkylene group of 1 to 3 carbonatoms or C═O, and p are 0 or 1, R₁ and R₂ are each alkyl groupscontaining 1 to 3 carbon atoms or are joined together to form ahetero-ring with the nitrogen atom and n is 1, 2 or 3.Pharmaceutically-acceptable salts of the diphenyl compounds may beemployed.

Alternatively, the benzene rings may be joined to form a tricyclic ring,in accordance with the structure: ##STR2##

In one preferred embodiment, the group ##STR3## is a diethylamino group,although other alkylamino groups may be employed, such as dimethylamino,and, in another preferred embodiment, a morpholino group, although otherheterocyclic ring groups may be employed, such as piperazino. o and pare usually 0 when Z is an alkylene group and n may be 2. In oneparticularly preferred embodiment, Z is --CH₂ --, n is 2, o and p areeach 0 and ##STR4## is a diethylamino group. This compound, namelyN,N-diethyl-2-[4-(phenylmethyl)-phenoxy]ethanamine, in the form of itshydrochloride salt, is abbreviated herein as DPPE. In addition to amethyl group linking the benzene rings, other linking groups may beemployed, such as ═C═O. Other substitutions may be made on the benzenerings in addition to the halogen atoms, for example, an imidazole group.

The compounds used herein are potent antagonists of intracellularhistamine binding at a site designated H_(IC). Such compounds, in anintracellular histamine binding assay, generally exhibit pki values ofat least about 5, preferably at least about 5.5. For example, DPPEexhibits a pki value of 6.5. L-histidinol used in the Warrington workreferred to above, is not as selective for intracellular histaminereceptors and in this regard, possesses a pki value of 3.1. Althoughweak affinity for binding to H_(IC) is exhibited by L-histidinol, thiscompound binds more strongly to other histamine binding sites, includingH₂ and H₃. The present invention employs compounds which potently andpreferentially bind to H_(IC).

In the present invention, significantly-smaller quantities of theantagonist compound are used in humans when compared to L-histidinol(typically 6 mg/kg or 240 mg/M² vs. 1000 mg/kg or 4000 mg/M²) and theantagonist compound is administered in the present invention over a muchshorter period before the chemotherapeutic agent(s) when compared toL-histidinol (typically 60 minutes vs. 5 hours).

The antagonist compound employed in the present invention isadministered to the patient in any convenient manner, such as byintravenous injection of a solution thereof in an aqueouspharmaceutically-acceptable vehicle.

The antagonist compound is administered to the patient over a suitableperiod of time before administration of at least one chemotherapeuticagent. The chemotherapeutic agent or a mixture of such agents may beadministered in any convenient manner consistent with its normal mannerof administration following conventional chemotherapeutic practice,often by intravenous infusion of a solution thereof. Suchchemotherapeutic agent solution may also contain the antagonistcompound. The invention is widely applicable to any type of knownanti-cancer drug, which tend to be compounds specific for treatment ofone type of cancer. A number of specific examples of such anti-cancerdrugs appear in the illustrated experimental data below.

The administration of the antagonist compound to the patient prior toadministration of the chemotherapeutic agent is necessary in order topermit the antagonist to inhibit the binding of intracellular histaminein normal and malignant cells and thereby, in effect, shut downproliferation of the normal cells, but increase proliferation ofmalignant cells.

The length of time prior to administration of the chemotherapeutic agentthat the antagonist compound is administered depends on the antagonistcompound, its mode of administration and the size of the patient.Generally, the antagonist compound is administered to the patient forabout 30 to about 90 minutes, preferably about 60 minutes, prior toadministration of the at least one chemotherapeutic agent.

The quantity of antagonist compound administered to the patient dependson the side effects to be ameliorated, but should be at least sufficientto inhibit binding of intracellular histamine in normal cells. Thequantity required to achieve the beneficial effects of the presentinvention depends upon the antagonist compound employed, thechemotherapeutic agent employed and the quantity of such agent employed.

In general, the quantity of antagonist compound employed in humans isfrom about 8 to about 320 mg/M² of animal to which the antagonistcompound is administered, with about 8 and 240 mg/M² being the optimaldose for gastro-intestinal and bone marrow protection, respectively.Over this dose range, the present invention is able to achieve anenhanced chemotherapeutic effect on cancer cells while, at the sametime, also protecting normal cells from damage by the chemotherapeuticagent in a wide variety of circumstances where traditional chemotherapyleads to damage of normal cells or tissues not involved in the diseaseprocess. Examples of the most common adverse effects on normal cellswhich result in traditional chemotherapy include:

(a) the killing of, or damage to, bone marrow cells, and

(b) the killing of, or damage to, normal cells lining thegastrointestinal tract.

In cancer-bearing animals, DPPE treatment alone modulates tumor growthwith promotion at lower doses and inhibition (cytotoxicity) at higherdoses. However, when combined with known anti-cancer drugs in the mannerdescribed herein, a marked synergistic action is observed whereby tumorsare inhibited or killed by the anti-cancer drugs. This effect has led,for example, to marked regressions or cures in some animal, includinghuman, cancers, such as sarcoma and melanoma.

As noted above, continued administration of the antagonist compoundfollowing administration of the chemotherapeutic agent, specifically upto about 30 mg/M² of DPPE on a daily basis, at least ameliorates, andoften eliminates, the side effects often associated with chemotherapy,including nausea, vomiting, anorexia and stomatitis, and preferably iseffected herein, with the longer the period of administration, the moresignificant is the protection against the side effects. A daily dose ofabout 240 to about 1200 mg/M² of DPPE affords maximum bone marrowprotection and synergy with chemotherapy to kill cancer cells.

Such continued administration of antagonist component is mostconveniently effected by intravenous administration, although oraladministration at the lower dose range may be feasible and, in somecases, more desirable from the standpoint of patient acceptance and ofdecreasing the load on the medical facility.

It has also been found that DPPE alone at low doses directly stimulatestumor cell growth in vivo and also increases the inflammatory responsein skin elicited by the tumor promoting phorbol ester, PMA (phorbolmyristate acetate). Several other classes of compounds, such asantidepressants, phenothiazines, triphenylethylene estrogens, histamine(H₁, H₂, H₃) antagonists, serotonin (5HT₁, 5HT₃) antagonists,β-andrenergic antagonists and imidazole analogs, also have beenidentified as producing the same results as observed for DPPE.

It now also has been found that tricyclic antidepressant drugs and thenon-tricyclic agent, fluoxetine (Prozac™), as well as H₁ -antihistamineand β-adrenergic antagonists, also compete for the binding of ³ H-DPPEand ³ H-histamine to H_(IC) in rat liver microsomes or brain membranesand, likewise, promote tumor growth.

Such compounds mimic the profiles of DPPE to inhibit normal cellproliferation but to promote malignant cell proliferation. Accordingly,these materials, at the proper dose level, could be predicted toincrease the therapeutic index of chemotherapy drugs and are includedwithin the scope of this invention.

Accordingly, in another aspect, the present invention provides a methodfor the treatment of cancer wherein a compound which inhibits normalcell proliferation while promoting malignant cell proliferation iscombined with chemotherapy agents to increase the therapeutic index ofchemotherapy drugs, in like manner to DPPE and similar potentantagonists of intracellular histamine binding as specifically describedherein.

Among the various compounds which may be employed in this aspect of thepresent invention are included:

(a) tricyclic antidepressants, such as amitriptyline, clomipramine andimipramine,

(b) non-tricyclic depressants, such as fluoxetine,

(c) phenothiazines, such as prochloroperazine, trifluoroperizine andchlorpromazine,

(d) H₁ antagonists, such as loratadine, hydroxyzine, phenyltoloxamineand astemizole,

(e) β-adrenergic agonists and antagonists, such as propanolol,

(f) serotonin (5HT₁ or 5HT₃) antagonists, such as ondansertron (5HT₃)and cyproheptadine (5HT₁),

(g) imidazoles and imidazole-like compounds, including H₂ antagonists,such as cimetidine and ranitidine, H₃ antagonists, such as thioperamideand antifungal agents, such as ketoconazole, and

(h) triphenylethylene derivatives, such as tamoxifen.

In general, the compounds which may be employed in this aspect of theinvention may have a chemical structure consisting of at least twophenyl rings, linked by a rigid third phenyl or non-phenyl ring, or by anon-rigid methyl, oxygen or other moiety, with the phenyl ring structurebeing linked by an ether, sulfhydryl or other ring structure or group toa basic alkylamine, imidazole or amino-imidazole side chain, forexample, the carboxyamide-amino-imidazole L651582.

Although this wide range of compounds may be employed to increase thetherapeutic index of chemotherapy drugs, DPPE and its direct analogs,may be significantly better agents for combination with chemotherapythan the foregoing groups of compounds, since DPPE appears to be morepotent and selective for H_(IC) and does not interact with calmodulin,protein kinase C or calcium channels and is only a weak antagonist atother common receptors, such as H₁, 5HT and D₂.

For example, DPPE does not cause serious toxic effects in humans atclinically relevant doses to enhance chemotherapy, whereas, for example,at their relevant concentrations to antagonize H_(IC), theantidepressant group of drugs and histidinol may cause cardiacarrythmias, H₁ antagonists might cause marked sedation, heart block orconvulsions and phenothiazines may cause dyskenesias.

THEORY

While the applicant does not wish to be bound by any theory to explainthe beneficial effects achieved by the present invention, the followingtheory is proposed. The compound administered to the patient is apotent, selective antagonist of histamine binding at a newly-discoverednovel intracellular receptor (H_(IC)) (see, for example, "Histamine asan Intracellular Messenger" by Brandes et al, Biochem. Pharmacol., vol40, 1990, pp 1677-1681). Intracellular histamine normally functionsthrough this receptor to mediate or modulate many important cellfunctions, including cell proliferation, immune responses and plateletaggregation.

Protection of the normal cells is achieved through antagonism ofhistamine at H_(IC) by the antagonist. Such antagonism results in atemporary complete shut-down of cell division, so that normal cells arenot susceptible to DNA damage in the presence of the chemotherapeuticagent(s), which preferentially attack dividing cells. In this way, forexample, DPPE is effective to block therapy-associated toxicity ofnormal bone marrow stem cells.

In addition, the antagonism results in an increase in the levels ofprostaglandins (natural substances which are known to protect tissuesfrom various injurious agents) in the tissue. For example, DPPEtreatment results in an increase by 500% in prostaglandin (PG)I₂, aprotective prostaglandin, in the gut. Through this mechanism, DPPE isknown to completely block ulcer formation in the presence of noxiousagents, such as alcohol and cysteamine (see U.S. Pat. No. 4,829,068 inwhich I am a co-inventor).

The antagonist further effects a potent blockage of the degranulation oftissue mast cells, whose granular contents, including histamine itself,have been linked to tissue damage and severe systemic side effects.Certain anti-cancer drugs, such as adriamycin, cause significant mastcell degranulation, an effect which has been related to cardiotoxiceffects.

As with bone marrow cells, the treatment of normal proliferatinglymphocytes (immune cells), according to the invention, results in adose-dependent blocking of DNA synthesis and a shut-down of these cellswithout causing cytotoxicity. The antagonist has an effect on bothT-lymphocytes and B-lymphocytes in the immune system. For example, DPPEis able to completely antagonize proliferation of T-lymphocytes in thepresence of Concanavalin A, a potent mitogen and plant lectin. DPPE alsoblocks the stimulation of antibody formation by the mediatorinterleukin-2 in certain B cells, resulting is a decrease in antibodyformation.

In contrast to its cytoprotective effect on normal cells and tissue invivo, as described herein, DPPE treatment stimulates, damages and/orkills malignant cells in vitro, depending on the dose, as described inU.S. Pat. No. 4,803,227, or those which are virally infected.

EXAMPLES Example I

This Example illustrates in vivo augmentation by DPPE of adriamycinanti-tumor activity in a murine sarcoma model.

C-3 fibrosarcoma cells (3×10⁵) were injected into the left glutealregion of C3H mice on day 0. On day 1, the mice were provided withtreatment by a combination of DPPE and adriamycin, administeredintraperitoneally. The DPPE was administered 60 minutes prior toadministration of the adriamycin. Mice also were administered withsaline, DPPE alone and adriamycin alone.

Animals in the experiments (n=12 for each group) were followed for 60days. At the end of the experimental period, those animals free ofpalpable tumors were considered cured.

The results obtained are set forth in the following Table I:

                  TABLE I                                                         ______________________________________                                                              Number of Rats                                          Treatment             Tumor-Free (n = 12)                                     ______________________________________                                        Saline                1                                                       Adriamycin (2 mg/kg)  0                                                       DPPE (50 mg/kg)       0                                                       DPPE (2 mg/kg)/Adriamycin (2 mg/kg)                                                                 1                                                       DPPE (25 mg/kg)/Adriamycin (2 mg/kg)                                                                3                                                       DPPE (50 mg/kg)/Adriamycin (2 mg/kg)                                                                7                                                       ______________________________________                                    

It will be seen from the results set forth in the above Table I, that,when adriamycin and DPPE alone are administered, no effect was obtainedwhereas when increasing quantities of DPPE were employed in combinationwith a constant quantity of adriamycin, an increased anti-tumor activitywas observed, such that, at the highest dose of DPPE tested (50 mg/kg),7 out of the 12 animals were cured.

Example II

This Example illustrates protection of bone marrow progenitors by DPPEin mice treated with a lethal dose of 5FU and adriamycin.

Mice of the strain C57B1 were administered a lethal dose (7.5 mg) of 5FU(5-fluorouracil), DPPE (100 mg/kg) or a combination of a lethal dose of5FU and DPPE (100 mg/kg or 4 mg/kg) and the results were compared with acontrol group to which saline only was administered. The DPPE and salinewere administered immediately prior to the 5FU. Bone marrow cell countswere made at 24 hours and 48 hours post administration.

The results obtained are set forth in the following Table IIA:

                  TABLE IIA                                                       ______________________________________                                                   CFU-C/                                                                        10.sup.4 cells.sup.(1)                                                                    R.C.S..sup.(2)                                         Treatment    24 h   48 h       24 h 48 h                                      ______________________________________                                        Saline       38.3   40.3       1.0  1.0                                       5FU          0.2    0.09       0.006                                                                              0.002                                     DPPE         36.7   38.6       0.96 0.96                                      DPPE                                                                          (100 mg/kg) +                                                                              35.7   37.3       0.93 0.93                                      5FU                                                                           DPPE                                                                          (4 mg/kg) +  38.3   33.3       1.0  0.83                                      5FU                                                                           ______________________________________                                         Note:                                                                         .sup.(1) No. of bone marrow colonies. CFUC = colonyforming units in           culture                                                                       .sup.(2) Relative Cell Survival                                          

Experiments paralleling those described above with 5FU were carried outemploying a lethal dose of adriamycin (20 mg/kg). The results obtainedfrom these experiments are set forth in the following Table IIB:

                  TABLE IIB                                                       ______________________________________                                                   CFU-C/                                                                        10.sup.4 cells                                                                            R.C.S.                                                 Treatment    24 h   48 h       24 h 48 h                                      ______________________________________                                        Saline       43.7   42.7       1.0  1.0                                       Adriamycin   1.8    0.65       0.04 0.006                                     DPPE (4 mg/kg)                                                                             42.3   41.3       0.97 0.97                                      DPPE         39.7   39.0       0.91 0.91                                      (4 mg/kg)/                                                                    Adriamycin                                                                    DPPE         43.3   41.3       0.99 0.97                                      (100 mg/kg)/                                                                  Adriamycin                                                                    ______________________________________                                    

As may be seen from the results set forth in the above Tables IIA andIIB, the administration of the DPPE along with the 5FU or adriamycinprovided almost complete protection for bone marrow progenitors from thelethal effects of the 5FU or adriamycin.

Example III

This Example illustrates in vivo augmentation of BCNU anti-tumoractivity in a B16 melanoma lung metastasis model.

5×10⁴ B16 melanoma cells were injected intravenously into the tail veinof C57B1 mice at day 0. The mice were treated with either saline, 32mg/kg of DPPE, 1 mg of BCNU or a combination of 32 mg/kg of DPPE and 1mg of BCNU, by intraperitoneal injection on day 1. The DPPE wasadministered 60 minutes before the BCNU.

In each group, six of the twelve animals were sacrificed at day 14 andlungs were removed for determination of metastasis. The remaining sixanimals were followed to death. The numbers and size of the lungmetastases were determined by visual or microscope count.

The results obtained are set forth in the following Table III:

                  TABLE III                                                       ______________________________________                                                  Numbers and              Median                                               (% control) of                                                                             Size of lung                                                                              Survival                                   Treatment lung metastases                                                                            metastases (1)                                                                            (days)                                     ______________________________________                                        Saline    241     --       All Macro 19                                       DPPE      219     (91%)    All Macro 21                                       BCNU      144     (60%)    All Macro 24                                       DPPE/BCNU  58     (27%)    All Micro 32                                       ______________________________________                                         (1) Macro means visually determined. Micro means microscopically only.   

As may be seen from the results in Table III, the inhibitory effect ofBCNU on the lung tumors was significantly increased by the additionalpresence of DPPE, which itself alone had a marginal effect.

Example IV

This Example illustrates in vivo augmentation of daunorubicin anti-tumoractivity in a B16 melanoma lung metastasis model.

The procedure of Example III was repeated employing daunorubicin inplace of BCNU. Groups of six mice were injected with B16f10 melanomacells and 24 hours later received saline, 4 mg/kg of DPPE alone, anon-lethal dose of daunorubicin alone (12.5 mg/kg) or DPPE (4, 25 or 50mg/kg) one hour prior to daunorubicin (12.5 mg/kg). All animals werefollowed to death or for 60-days post injection, and sacrificed for lungmetastases.

The results obtained are set forth in the following Table IV:

                  TABLE IV                                                        ______________________________________                                                        Median-Survival                                                                             No. Cures                                       Treatment Group (days)        (n = 6)                                         ______________________________________                                        Saline          17            0                                               Daunorubicin (12.5 mg/kg)                                                                     25            0                                               DPPE (4 mg/kg) +                                                                              29            2                                               Daunorubicin (12.5 mg/kg)                                                     DPPE (25 mg/kg) +                                                                             .sup. 60.sup.+                                                                              4                                               Daunorubicin (12.5 mg/kg)                                                     DPPE (50 mg/kg) +                                                                             .sup. 60.sup.+                                                                              4                                               Daunorubicin (12.5 mg/kg)                                                     ______________________________________                                    

As may be seen from the results of Table IV, the inhibitory effect ofdaunorubicin or lung tumors was enhanced by the presence of DPPE.

Example V

This Example shows in vivo host cytoprotection from a lethal dose ofadriamycin.

Saline or DPPE (2 mg/kg) were administered to DBA/2 mice 1 hour (n=12)or 15 minutes (n=6) prior to administration of 15 mg/kg of adriamycin.The number of survivors after 30 days was determined. The results areset forth in the following Table V:

                  TABLE V                                                         ______________________________________                                        Treatment        Number of Survivors                                          ______________________________________                                        Saline            4/12 (33%)                                                  DPPE             13/18 (72%)                                                  ______________________________________                                    

As may be seen from Table V, the administration of DPPE provided in vivohost cytoprotection from the lethal dose of adriamycin.

Example VI

This Example illustrates the effect of DPPE on thymidine incorporationinto lymphocyte DNA.

Spleen cells from BALB/C mice were stimulated with Concanavalin A (5μg/ml). The cells then were treated with varying does of DPPE and thelevel of thymidine incorporated into DNA was determined. The resultswere plotted graphically and appear as FIG. 1. As may be seen from thisFigure, at a dosage level of 25 μM, DPPE completely blocks thymidineincorporation into DNA but does not adversely affect cell survival.Thus, the DPPE treatment puts normal proliferating lymphocytes into astate of growth arrest without causing cytotoxicity.

The experiment was repeated using 2.5 μg/ml of Concanavalin A in placeof 5 μg/ml and 2% fetal calf serum in place of 10%. The results obtainedare illustrated in FIG. 2. At concentrations of DPPE which inhibited DNAsynthesis (5 μm), no significant cytotoxicity was observed.

Example VII

This Example illustrates the effect of DPPE on thymidine incorporationinto DNA in transformed lymphocytes.

The experiment of Example VI was repeated employing virally-infectednon-senescing transformed spleen-derived lymphocytes (S-10) also ofBALB/C origin with 0.25 nM of ³ H-thymidine added. The results wereplotted graphically and appear as FIGS. 3 and 4 respectively. As may besee therein, in contrast to FIGS. 1 and 2, 25 μM of DPPE causedapproximately 50% cytotoxicity to the virally-infected cells. Whenadjusted for cell number, thymidine incorporation increased at cytotoxicconcentrations of DPPE (10 to 25 μM).

The experiment was again repeated using human breast cancer cell (MCF-7)with 0.25 nM of thymidine added. The results were plotted graphicallyand appear as FIG. 5. Analogous results can be seen to those observedwith the S-10 cells.

Example VIII

A clinical study was carried out in 24 patients with advanced cancer.

(a) DPPE alone was administered to patients to determine a safe doserange in humans. The highest non-toxic dosage was found to be 4 mg/kg(160 mg/M²) given intravenously over a one-hour period. At 6 mg/kg (240mg/M²) over one hour, CNS toxicity (as manifested by any or all ofmuscle twitching, a drop of 1° to 2° C. in body core temperature,auditory hyperacusis or hallucination, choreoathetosis, cerebellarataxia and projectile vomiting) was observed. However, significant CNStoxicity was absent when 6 mg/kg (240 mg/M²) was administered IV over 2hours, suggesting that peak serum level determines CNS toxicity. Whenthe dose is converted to mg/M², threshold for CNS toxicity occurs at thesame dose previously observed in preclinical toxicology (ip route)studies in mice (240 mg/M²).

(b) DPPE at a daily dose of 0.2 mg/kg (8 mg/M²), given as an IV infusionover 24 to 72 hours was found to be entirely without clinical sideeffects, with the possible exception of constipation in occasionalpatients, and not to cause any significant changes in biochemistries orblood counts. This dose of DPPE also has been determined to potentlyprevent, or ameliorate by over 90%, nausea, vomiting, anorexia andstomatitis in 58 patient treatments with Adriamycin (60 mg/M²). GIprotection was most pronounced when DPPE was given at a dose of 0.2mg/kg (8 mg/M²) daily for 72 hours post-treatment by IV infusion.

(c) Higher single IV doses of DPPE (1, 2, 4 mg/kg) given over 1 houralso appear to be significantly antiemetic against Adriamycin, althoughsome patients experienced nausea, or transient anorexia, at 4 mg/kg (160mg/M²) of DPPE alone. At doses of 1 to 6 mg/kg IV over 1 hour, DPPEalone also was found to cause a transient decrease (20 to 30%) inneutrophil counts in 4/6 patients, with complete recovery by day 5 to 7.No significant effect of DPPE alone on platelets, hemoglobin orbiochemistry has been observed.

(d) Using 0.2 mg/kg (8 mg/M²) of DPPE as a total daily dose, increasedduration of treatment improved the therapeutic benefit to preventnausea, vomiting, anorexia and a drop in nadir white counts, but notalopecia, caused by Adriamycin at a dosage level of 60 mg/M². A 24-hourDPPE infusion was an effective anti-emetic therapy in the first 24 to 48hours following Adriamycin administration, but many patients thenexperienced delayed nausea, vomiting and/or anorexia at 72 or 96 hoursafter Adriamycin administration. However, when given as a 72-hourinfusion, DPPE was observed to block completely all acute and delayedgastrointestinal side effects of Adriamycin in four patients. Inaddition, two additional patients experienced only one minor episode ofnausea and/or vomiting in the first 24 hours following Adriamycinadministration, but then were well without any need for antiemetics. The72-hour infusion of low dose DPPE also prevented mouth ulceration in onepatient who had previously experienced this symptom during all previousnon-Adriamycin chemotherapy and DPPE/Adriamycin given in shorterschedules.

(e) As compared to other regimens of DPPE/Adriamycin, nadirpolymorphonuclear WBC counts at 14 days appear to be highest in patientswho received 0.2 mg/kg (8 mg/M²) DPPE for 72 hours (1,285±385;mean±S.E.M.). Platelet counts have been uniformly above 150,000/mm³ atDay 14.

(f) In seventeen evaluable patients, seven major responses, includingthree breast cancers, one lymphoma, one rhabdomyosarerma and onemedullary carcinoma of thyroid, have been documented.

Example IX

In a second human pilot study, the infusion of higher doses of DPPE (2to 12 mg/kg; optimal dose=6 mg/kg (240 mg/M²)) over a period of 60minutes prior to the administration of an antineoplastic drug resultedin significant palliation in 16/20 evaluable patients with progressivemetastatic cancer, refractory to chemotherapy alone (1 complete and 6partial remissions, 5 improved, 4 stabilized, 4 progressed). Manypatients remaining on treatment (the longest for 11+ months) continue toimprove, or be stable, suggesting a durable response, and thatadditional complete or partial remissions may result in some of thesesubjects.

The most impressive responses have been observed in patients with breastand colon cancer, and lymphoma. Treatment with DPPE (240 mg/M²) plus5-fluorouracil (750 mg/M₋₋ ²) once weekly in 6 patients with breastcancer has resulted in resolution of liver metastases in two patients,progressive healing of bone metastases in one patient and significantresolution of chest wall metastases in two patients. Similarly, fourpatients on DPPE plus 5-FU for colon cancer with liver metastases,previously unresponsive to 5-FU plus leucovorin, have shown significantimprovement of liver lesions and/or liver function and otherdisease-associated symptoms such as pain; a fifth patient with tumorinvading the bladder, has had resolution for the past four months ofgross daily hematuria. Protection of bone marrow stem cells has beenobserved in several patients treated with DPPE and high doses ofcyclophosphamide (1 g/M²). Thus, as in pre-clinical studies in mice,high doses of DPPE maximally synergize with chemotherapy to kill cancercells, while significantly protecting the bone marrow from themyelosuppressive effects of chemotherapy.

Example X

This Example illustrates the tumor promoting and pro-inflammatoryresponse effects of DPPE alone.

FIG. 6 shows the tumor-promoting effect DPPE (1 mg/kg or 4 mg/M²) givensubcutaneously once daily×3, to seven DBA/2 mice inoculatedsubcutaneously with 2×10² L5178Y lymphoma cells 48 hours previously. Asecond group of 7 tumor cell-inoculated mice served as controls (salineinjections, once daily×3). By day 14, 7/7 DPPE treated animals hadpalpable tumors as compared to 4/7 controls. At the end of 4 weeks, 6/7controls had tumors with an aggregate surface area of 14.5 cm²(mean=2.1±0.8 cm² /animal), while 7/7 DPPE-treated animals had tumorswith an aggregate surface area of 38.4 cm² (mean=5.5±0.7 cm² /animal).Thus, the tumor burden of DPPE-treated animals was approximately2.5-fold greater than that of controls.

To investigate any effect of DPPE to increase PMA-induced inflammationin the same strain of mice (DBA/2), groups of 3 animals were shaved overthe back and 48 hours later received a single topical application of 17nM PMA in acetone. The PMA-treated mice then received either saline(control) or DPPE (4 or 32 mg/kg at time 0 and 24 hours). Three animalspainted with acetone served as vehicle controls. Forty-eight hourslater, the various groups were sacrificed by CO₂ asphyxiation, the skincarefully excised, pinned to paper strips to prevent wrinkling, andimmersed in formaldehyde. H and E-stained sections of skin were assessedfor degree of inflammation.

It was observed that the animals who received DPPE had a significantlygreater inflammatory response to PMA as compared to saline or acetonecontrols. The most intense inflammatory response was seen in animalsreceiving the high dose (32 mg/kg or 128 mg/M²) of DPPE, where increasedmitotic activity in the epithelial layer was also noted as compared tothe PMA and saline-treated groups. The results of the experimentsreported in this Example clearly show that DPPE enhances theinflammatory response of the tumor promoter PMA. Indeed, since tumorpromotion requires the presence of inflammatory response, and can beblocked by agents which inhibit inflammation by definition, DPPEfunctions as a co-promoter with PMA.

Example XI

This Example shows the H_(IC) binding and tumor promoting effects ofcertain compounds and the antiproliferative effect of DPPE and certaincompounds.

FIG. 7 shows the potency of two tricyclic agents, namely amitriptylineand doxepin, to compete for ³ H-DPPE binding in liver microsomes. TheK_(d) value for DPPE is 65 nM while the K_(i) for doxepin is 5 μM andfor amitriptyline is 10 μM. Doxepin and fluoxetine also compete for ³H-histamine binding to H_(IC) in brain membranes (K_(f) =10 μM; FIG. 8).

FIG. 9 demonstrates the tumor-promoting effects of the tricyclic agent,amitriptyline, and the non-tricyclic agent, fluoxetine, in C3H miceinjected subcutaneously into the gluteal region with 1×10⁵ C-3fibrosarcoma cells. The doses employed were equivalent to therapeutichuman doses (80 mg/M² for amitriptyline and 20-40 mg/M² for fluoxetine).The experiments were blinded so that the individual measuring the firstappearance of palpable tumor was unaware of the treatment group (salinecontrol vs antidepressant drug; n=10 in each group).

It may be seen from this data that, in both experiments, the controlanimals did not develop tumors until day 6, whereas in thefluoxetine-treated animals, tumors appeared on days 3, 4 and 5post-injection and, in the amitriptyline-treated animals, tumorsappeared on days 4 and 5 post-injection. Thus, in both experiments, 4/10of antidepressant-treated animals, but no controls had tumors by day 5(8/20 vs 0/20 controls, both experiments combined).

Conversely, FIG. 10 shows that, like DPPE, both amitriptyline andfluoxetine inhibit, in the absence of cytotoxicity, the proliferation ofconcanavalin A-stimulated normal lymphocytes (IC₅₀ =10 to 20 μM). Thus,although weaker than DPPE, these agents inhibit the proliferation ofnormal stem cells while increasing the proliferation of tumor cells.

FIG. 11A shows that propanolol (a β-adrenergic antagonist) inhibitshistamine binding to H_(IC) in microsomes and FIG. 11B shows thatpropanolol inhibits normal lymphocyte mitogenesis. In a C-3 fibrosarcomamurine model, propanolol significantly increased tumor weight on Day 23,as seen in FIG. 12. Similarly loratidine (a tricyclic non-sedating H₁-antihistamine) potently promoted tumor growth, as seen in FIG. 12 andalso inhibited concanavalin A-stimulated mitogenesis (FIG. 13).Astemizole (a non-sedating H₁ -antihistamine) similarly is potent toinhibit histamine binding and concanavalin A-stimulated mitogenesis(data not shown) and, in two separate experiments, to potently stimulatethe growth of C-3 fibrosarcoma, as shown in FIG. 14.

The compounds for which binding and proliferation data are provided inthis Example, therefore, mimic the profiles of DPPE to inhibit normalcell proliferation but to promote malignant cell proliferation (ExampleIV). On the basis of his profile, these agents, at the proper doselevel, may be predicted to increase the therapeutic index ofchemotherapy drugs.

SUMMARY OF DISCLOSURE

In summary of this disclosure, the present invention provides a novelapproach to chemotherapeutic treatment of cancer whereby an enhanced invivo effect of the chemotherapeutic agent is obtained while achievingprotection of normal cells, particularly bone marrow andgastro-intestinal cells, from the toxic effects of the chemotherapeuticagent. Modifications are possible within the scope of this invention.

What I claim is:
 1. A method of protecting normal bone marrow cells inan animal from the adverse effects of chemotherapy, whichcomprises:administering to said animal at least one diphenyl compound ofthe formula: ##STR5## wherein X and Y are each fluorine, chlorine orbromine, Z is an alkylene group of about 1 to 3 carbon atoms or ═C═O, orthe phenyl groups are joined to form a tricyclic ring, o and p are 0 or1, R₁ and R₂ are each groups containing 1 to 3 carbon atoms or arejoined together to form a hetero-ring with the nitrogen atom and n is 1,2 or 3, in an amount of from about 8 to about 240 mg/M² of animal forabout 60 to about 90 minutes prior to commencement of said chemotherapy.2. The method of claim 1 wherein, following said chemotherapy, saiddiphenyl compound is administered in a daily amount of from about 30 toabout 1200 mg/M² of animal, so as to ameliorate adverse side effects ofsaid chemotherapy.
 3. A method for protecting normal cells lining thegastrointestinal tract from the adverse effects of chemotherapy, whichcomprises:administering to said animal at least one diphenyl compound ofthe formula: ##STR6## wherein X and Y are each fluorine, chlorine orbromine, Z is an alkylene group of about 1 to 3 carbon atoms or ═C═O, orthe groups are joined to form a tricyclic ring, o and p are 0 or 1, R₁and R₂ are each groups containing 1 to 3 carbon atoms or are joinedtogether to form a hetero-ring with the nitrogen atom and n is 1, 2 or3, in an amount of from about 8 to about 30 mg/M² of animal for about 30to about 90 minutes prior to commencement of said chemotherapy.
 4. Themethod of claim 3 wherein, following said chemotherapy, said diphenylcompound is administered in a daily amount of from about 8 to about 12mg/kg of animal, so as to ameliorate adverse side effects of saidchemotherapy.
 5. The method claimed in claim 1 or 3 wherein said atleast one diphenyl compound is DPPE.
 6. The method of claim 5 whereinsaid chemotherapy is effected using adriamycin.